Chromogenic compounds are indicative compounds capable of producing color by causing a displacement of, or the appearance of, absorbent bands in the visible spectrum. Indicative compounds are useful because they have significant applications in the fields of biotechnology, diagnostic chemistry, microbiology, molecular biology and the like.
Indicative compounds are, among other things, particularly useful in the identification of biological material such as genetically transfected cells and organisms. Their employment in cloning vectors, which are used to create genetically engineered cells and organisms, is well known. Specifically, a histochemical marker gene may be incorporated into a cloning vector in order to subsequently identify successful transfectants. A successful transfectant carries both the desired gene and the histochemical marker gene. The marker gene may be induced to express a marker gene product in the form of a marker enzyme. Thereafter, a chromogenic compound may be used to detect the presence of a particular marker enzyme by generating distinct colored reaction products as a result of enzymatic activity.
Likewise, the use of multiple histochemical marker genes, which generate different color reaction products, would facilitate the analysis of multiple cell populations. In particular, the use of multiple histochemical marker genes permits the distinction between two or more cell types possessing histochemically different marker genes. Thus, it is desirable to develop techniques wherein transfectants, which carry different histochemical marker genes, may be distinguished from each other.
Indicative compounds are also useful with regard to locating a specific sequence of DNA or RNA. In particular, specific sequences of DNA or RNA may be located on chromosomes or other genetic material with the use of nucleic acid probes. Nucleic acid probes contain short segments of nucleic acids which are complimentary to the specific DNA or RNA sequence to be located. Currently, it is well known to prepare nucleic acid probes containing radioactive isotopes, i.e., of phosphorus, in order to subsequently identify and locate the nucleic acid probe on a chromosome or on other genetic material. Therefore, for environmental and safety reasons, it is desirable to develop alternative, non-isotopic techniques which locate and identify nucleic acid probes in order to eliminate the use of dangerous radioactive materials.
Furthermore, indicative compounds are useful when used in conjunction with enzyme-antibody conjugates. Enzyme-antibody conjugates are important with respect to enzyme linked immunosorbent assays. In this regard, enzymes are conjugated with antigen specific antibodies which permit detection of a particular antigen. Thus, in order to facilitate the detection of a particular antigen, it is desirable to improve methods which utilize enzyme-antibody conjugates, such as immunoblotting techniques.
In microbiology, the presence of indicator organisms is frequently used to determine the quality of various products. For example, in the analysis of water, food and dairy products, the presence of members of the "coliform" group as well as the presence of the bacterial species Escherichia coli (E. coli) are considered very significant quality indicators. Numerous methods for determining, identifying and enumerating indicator organisms currently exist, with varying degrees of accuracy and facility. Some test methods merely indicate the presence or absence of the organisms whereas other methods attempt to quantify the organisms in the test materials.
For instance, the reagent 5-bromo-4-chloro-3-indolyl-.beta.-D-galactopyranoside (X-Gal) is a known test compound for identifying coliforms. When acted on by the .beta.-galactosidase enzyme produced by coliforms, X-Gal forms an insoluble indigo blue precipitate. X-Gal can be incorporated into a nutrient medium such as an agar plate, and if a sample containing coliforms is present, the coliforms will grow as indigo blue colonies. Since X-Gal forms an insoluble precipitate, rather than a diffusible compound, X-Gal allows the quantitative determination of coliforms.
Recently, a similar compound, 5-bromo-4-chloro-3-indolyl-.beta.-D-glucuronic acid (X-Gluc) has been developed for the identification of E. coli. When acted on by the .beta.-glucuronidase enzyme produced by E. coli, X-Gluc forms an insoluble indigo blue precipitate. Since X-Gluc forms an insoluble precipitate, 15 rather than a diffusible compound, X-Gluc allows the quantitative determination of E. coli. Further, it does not require the use of ultraviolet light. X-Gluc and its ability to identify E. coli are described in Watkins, et al, Appl. Environ. Microbiol. 54:1874-1875 (1988). A similar compound, indoxyl-.beta.-D-glucuronide, which also produces sharp blue colonies of E. coli, was described in Ley, et al, Can. J. Microbiol. 34:690-693 (1987).
X-Gal and X-Gluc have the disadvantage that they each contain the exact same chromogen and therefore the two cannot be used together to identify and distinguish between both E. coli and general coliforms in a single test with a single sample. The two indicator compounds cannot be used together because both X-Gal and X-Gluc generate the formation of identically hued indigo blue colonies. On one hand, a person using both reagents together would be able to quantitatively identify the total number of coliforms, however; on the other hand, the reagents would not be able to indicate which of the colonies were E. coli and which were other coliforms besides E. coli. Therefore, it is desirable to develop novel indicator compounds which can identify and enumerate indicator organisms when used alone and in combination with other indicator compounds.
Accordingly, it is clear from the foregoing that improved methods to effectively quantify, identify and/or differentiate biological material such as microorganisms, transfectants, antigens, DNA or RNA sequences and the like are needed, and there is a continuing search for better, more accurate, simpler and varied methods in these areas.